News Physiol Sci 15: 171-175, 2000;
1548-9213/00 $5.00
News in Physiological Sciences, Vol. 15, No. 4, 171-175,
August 2000
© 2000 Int. Union Physiol. Sci./Am. Physiol. Soc.
Angiotensin II Receptor Physiology Using Gene Targeting
Michael I. Oliverio and
Thomas M. Coffman
M. I. Oliverio and T. M. Coffman are at Duke University and Durham Veterans Affairs Medical Centers, Room 1100/Building 6, 508 Fulton Street, Durham, North Carolina, 27705.
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Abstract
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The study of mice with targeted disruptions of angiotensin receptor genes has provided new insights into the roles of the individual receptor subtypes, i.e., AT1A, AT1B, and AT2, in growth, development, and the regulation of blood pressure.
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Introduction
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The renin-angiotensin system (RAS) is an important regulator of blood pressure and body fluid homeostasis. The effects of this system are primarily mediated by the multifunctional peptide angiotensin II, which is produced through a series of enzymatic reactions. Circulating angiotensinogen is made in the liver. The protease renin, which is released from the juxtaglomerular apparatus of the kidney, cleaves angiotensinogen to form the decapeptide angiotensin I, which is then further cleaved by angiotensin-converting enzyme (ACE) to form the octapeptide angiotensin II. The many biological effects of angiotensin II are mediated through its interaction with specific cell surface receptors.
The angiotensin receptors are divided pharmacologically into two types, type 1 (AT1) and type 2 (AT2), on the basis of differential binding to nonpeptide antagonists. Angiotensin receptors belong to the large family of rhodopsin-like G protein-coupled receptors and structurally possess seven transmembrane domains. AT1 receptors have been cloned from several species, and two subtypes, AT1A and AT1B, have been identified in rodents, whereas man possesses a single type of AT1 receptor. The 1A and 1B isoforms are the products of distinct genes located on separate chromosomes. The binding signatures of AT1A and AT1B receptors are virtually identical. AT1 receptors are expressed in kidney, heart, brain, adrenal gland, vascular smooth muscle, and several other tissues. In mice and rats, the AT1A subtype is the predominately expressed AT1 receptor in most of these tissues, except for the anterior pituitary gland and adrenal zona glomerulosa, where AT1B expression appears to be more prominent.
Angiotensin II regulates blood pressure through its actions as a potent vasoconstrictor, its ability to modulate sodium reabsorption by the kidney, and by stimulating the release of both vasopressin and aldosterone. Angiotensin II stimulates cellular proliferation in a number of cell types, and several studies have identified roles for angiotensin II in growth and development. These classically recognized functions of angiotensin II are primarily mediated by AT1 receptors.
AT2 receptors are widely expressed in fetal tissues during late gestation but are also present in adult rodents and humans in discrete locations within the brain, heart, and kidney. The signal transduction pathways of the AT2 receptor have not been as clearly defined as that of AT1 receptors. In general, AT2 receptors appear to have functions opposite (and perhaps balancing) those of AT1 receptors (6).
The RAS contributes to the pathogenesis of several human diseases, including hypertension, congestive heart failure, coronary artery disease, and diabetic nephropathy. Although the effects of angiotensin II on hemodynamic and cellular function both promote pathology, their relative importance and the specific contributions of individual receptor subtypes to cardiovascular disease is not clear. Thus elucidating the physiological function of angiotensin receptors should provide insights into normal physiology and into the pathogenesis and treatment of common human diseases.
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Gene targeting and the RAS
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Using homologous recombination in embryonic stem cells, genes can be altered in a specific and targeted fashion. This, in turn, allows the physiologist to examine the effects of altering a single gene product on the homeostasis of an intact animal. Gene targeting has been successfully applied to the study of the RAS, and all of the known genes that encode elements of this system have been disrupted. Because germ-line competent embryonic stem cells have only been isolated from mice, this small mammal is the only species in which gene targeting is currently possible. The body size of the mouse was an initial impediment to physiological experimentation. However, many techniques used in larger species have now been adapted for use in mice. In this review, we will discuss contributions of gene-targeting experiments to understanding the multiple functions of angiotensin receptors.
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Angiotensin receptors in growth and development
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Several lines of evidence have indicated important roles for angiotensin II in growth and development. RAS gene expression is enhanced in many fetal tissues, and angiotensin receptors are expressed in a tightly regulated pattern during late gestation and in the postnatal period. In rodents and sheep, treatment with ACE inhibitors or AT1 receptor antagonists, even for a limited time in the neonatal period, results in diminished somatic and kidney growth as well as abnormal kidney structure. The use of ACE inhibitors during pregnancy is also teratogenic to the human fetus and may result in oligohydramnios and neonatal renal failure.
This evidence supporting a role for the RAS in development suggested that dramatic phenotypes would be seen in the mice with targeted disruption of the individual RAS genes. Surprisingly, mice that are unable to synthesize angiotensin II due to targeted disruptions of either the angiotensinogen or ACE genes are born in expected numbers with normal structures of their major organs. However, a significant number of these angiotensin II-deficient mice die during the early postnatal period (1). Moreover, ACE- or angiotensinogen-deficient mice that survive to adulthood have diminished somatic growth and develop a series of structural abnormalities in their kidneys. The kidney pathology consists of medial arterial thickening of interlobular vessels and atrophy of the inner medulla. This vascular pathology is not present in other organs such as heart, adrenal gland, or liver. Because the phenotypes of ACE and angiotensinogen deficiency are virtually identical and the phenotype can be rescued in angiotensinogen-deficient mice by human renin and angiotensinogen transgenes, its pathogenesis seems to be caused by the absence of angiotensin II (2).
The role of individual angiotensin receptors in the abnormalities caused by angiotensin II deficiency was clarified by analysis of mice with targeted disruption of the AT1A, AT1B, and AT2 receptors. On mixed genetic backgrounds, the phenotype of reduced survival and kidney abnormalities was not recapitulated by the individual targeted disruption of the known angiotensin receptors (1). AT1A receptor-deficient mice have a minimal survival disadvantage that is evident only after analysis of large numbers of offspring, and they manifest hypertrophied juxtaglomerular apparati. However, they lack the obvious atrophy of the inner medulla seen in angiotensin II-deficient mice. Analysis of large numbers of AT1A knockouts also failed to demonstrate any deficits in renal, cardiac, or somatic growth. Using careful morphometric analysis, Matsusaka et. al. (9) found that the intrarenal arteries of AT1A knockouts exhibit mild medial thickening but to a much lesser degree than angiotensin II-deficient mice. The AT1B- and AT2-deficient mice have normal renal morphology and are visually and histomorphologically indistinguishable from their wild-type littermates.
Because the AT1A and AT1B genes are on separate chromosomes, AT1A receptor-deficient and AT1B receptor-deficient mice could be crossed, ultimately yielding mice with combined disruption of both AT1A and AT1B receptors that are completely deficient of AT1 receptors (12, 14). These AT1 receptor-deficient mice have diminished survival and somatic growth, resembling that seen in angiotensin II-deficient mice. AT1A/AT1B double knockout mice also develop the severe kidney phenotype of angiotensin II-deficient mice, as illustrated in Fig. 1
. Thus the lack of either individual AT1 isoform is compensated for by the remaining receptor in regard to these phenotypes. These studies demonstrate the importance of AT1 receptors in mediating the effects of angiotensin II in growth and development.
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FIGURE 1. Kidney histomorphology of wild-type and AT1A/AT1B angiotensin receptor-deficient mice. A and B: axial sections from wild-type (A) and AT1A/AT1B receptor-deficient mice (B). Magnification = x5. There is marked atrophy of the inner medulla in the AT1-deficient kidney compared with the control. C and D: cross-sections of representative small arteries in kidneys from wild-type (C) and AT1A/AT1B receptor-deficient mice (D). The media of the vessels from the mutant animals was markedly thickened, and many of the vessels are surrounded by inflammatory cells as shown. Magnification = x150. Figure reprinted from Ref. 12 with permission.
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Regulation of blood pressure and sodium balance
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The RAS is a potent regulator of sodium and fluid balance. Dysregulation of the RAS causes chronic elevations in blood pressure in experimental models and in patients with hypertension. The efficacy of antagonists of the RAS as antihypertensive agents in large numbers of affected individuals provides compelling evidence for the role of this system in the pathogenesis of hypertension. The Guyton hypothesis proposes that the actions of the RAS in causing hypertension must involve chronic alterations in renal sodium handling (4).
The role of the RAS in modulating sodium excretion by the kidney has been well documented. For example, in animal models and in humans, suppression of angiotensin II production is required for normal excretion of a sodium load, whereas chronic administration of subpressor doses of angiotensin II increases blood pressure by altering sodium balance (5). Conversely, during sodium depletion, pharmacological inhibition of the RAS causes an inappropriate natriuresis and reductions in blood pressure. Angiotensin II may affect sodium excretion through several discrete mechanisms, including: 1) effects on renal hemodynamics, 2) direct stimulation of renal tubular sodium reabsorption, and 3) stimulation of aldosterone production by the adrenal glands.
The severe structural abnormalities of the angiotensin II-deficient models (angiotensinogen or ACE knockout mice) limit their usefulness in the study of the physiological actions of angiotensin II in the kidney. Because kidney structure is normal in mice with targeted deletion of either AT1A or AT1B or of AT2 receptors, these lines of mice are ideal models to study the roles of angiotensin II in the regulation of blood pressure and sodium balance.
AT1 receptors.
Targeted disruption of the angiotensinogen, ACE, or AT1A receptor genes decreases blood pressure (1). The magnitude of blood pressure reduction, measured directly by intra-arterial catheters or indirectly using tail-cuff manometry, is similar in these three lines of knockout mice and is ~25–30 mmHg. Additionally, a more subtle alteration of angiotensinogen or AT1A receptor expression in mice heterozygous for the respective gene disruptions causes a modest but significant reduction of blood pressure, as shown in Fig. 2 (8). This indicates that mutations causing quantitative alterations in expression of these genes may have significant effects on resting blood pressure.
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FIGURE 2. Blood pressures measured using tail-cuff manometry (A) and intra-arterial catheters (B) in wild-type mice (+/+), mice heterozygous for targeted disruption of the AT1A receptor gene (+/–), and mice homozygous for targeted disruption of the AT1A receptor gene (–/–) *P = 0.011 vs. +/+; **P < 0.0001 vs. +/+ and P = 0.001 vs. +/–; §P = 0.03 vs. +/+; ¶P = 0.0003 vs. +/+ and P = 0.02 vs.+/–. Figure reprinted from Ref. 8 with permission.
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In contrast, AT1B receptor-deficient mice do not exhibit any reduction in blood pressure or any discernible phenotype. Therefore, targeted disruption of the AT1B receptor gene did not initially clarify its function. However, AT1A-deficient (Agtr1a–/–) mice provided direct evidence for actions of AT1B receptors in blood pressure regulation (1). AT1A receptor-deficient mice exhibit a modest pressor response to angiotensin II that is blocked by AT1 receptor antagonists but not by sympatholytic agents, suggesting direct vasoconstrictor actions of AT1B receptors. In addition, chronic treatment of AT1A receptor-deficient mice with an AT1-specific antagonist causes a further reduction in their systolic blood pressures, suggesting a role for AT1B receptors in the regulation of blood pressure when AT1A receptors are absent. Furthermore, AT1A/AT1B receptor double knockout mice have no detectable pressor response to angiotensin II (12, 14). These findings confirm the actions of AT1B receptor in producing vasoconstriction and argue strongly against the presence of other angiotensin receptors with acute vasoactive effects.
AT2 receptors.
The vasoconstrictor actions of angiotensin II are augmented in AT2 knockout mice, and at least one of the lines of mice has elevated blood pressure (6). These studies of AT2 receptor-deficient mice are consistent with other accumulating evidence suggesting that AT2 receptors act to negatively modulate the actions of AT1 receptors to raise blood pressure. Our experiments using AT1A/AT1B receptor-deficient mice also support this view (12). We found that treatment of wild-type mice with an ACE inhibitor causes a substantial reduction in blood pressure. In contrast, ACE inhibition causes a paradoxical increase in the blood pressure of AT1A/AT1B double knockouts. We speculate that this effect represents inhibition of AT2 receptor signaling. Furthermore, the absence of an acute vasodepressor response to angiotensin II infusions in the AT1A/AT1B double knockouts suggests that the effect of AT2 receptors in regulating blood pressure is related to modulation of renal sodium handling rather than direct vascular actions (12, 14). Recent experiments by Siragy et al. (13) suggest that the negative modulating actions of AT2 receptors may play a role in the pathogenesis of hypertension. These investigators found that AT2 receptor-deficient mice develop significant hypertension and reductions in sodium excretion during chronic infusion of low doses of angiotensin II that did not alter blood pressures in wild-type controls. The AT2 knockout mice had low basal levels of bradykinin and cGMP in renal interstitial fluid, indicating reduced nitric oxide production in the kidney. In wild-type mice, sodium restriction or angiotensin II infusion increased levels of bradykinin and cGMP in renal interstitial fluid; however, this response was absent in AT2 knockout mice. Thus, although the mechanism of regulation of blood pressure by the AT2 receptor remains to be clarified, available data suggests that the AT2 receptor modulates renal sodium handling, possibly through effects on nitric oxide production.
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Angiotensin receptors and water balance
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The RAS also regulates water metabolism. In the brain, angiotensin II stimulates thirst and can regulate vasopressin (antidiuretic hormone) release. Angiotensin II may also affect water handling in the kidney through its ability to regulate proximal nephron reabsorption of water and solute and by regulating blood flow in the inner medulla. Additionally, AT1 receptors are expressed in the thick ascending limb of Henle's loop and in collecting ducts, sites that play crucial roles in urinary concentration.
Both angiotensin II-deficient and AT1A/AT1B double knockout mice have defects in their ability to concentrate urine in response to thirsting (12). Given the atrophy of the renal inner medulla seen in these models, this finding was not completely unexpected. Miyazaki et al. (10) have demonstrated arrested development and diminished peristalsis of the renal pelvis in AT1A/AT1B double knockout mice. It was hypothesized that this abnormality causes an obstructive nephropathy with subsequent atrophy of the inner medulla of the kidney. However, AT1A receptor-deficient mice (12) that lack the severe atrophy of the inner medulla of AT1A/AT1B double knockouts and ACE- or angiotensin-deficient mice also have significant defects of urine concentration (Fig. 3). The same conclusion applies to mice that lack only the tissue-bound form of ACE (3). The defect in urinary concentration in AT1A knockout mice seems to be caused by a kidney defect since their vasopressin responses to thirsting are normal. Furthermore, a urine-concentrating defect is induced in wild-type mice after brief treatment with an AT1 receptor antagonist (11). Together, these findings demonstrate a physiological role for AT1A receptors in the regulation of water handling by the kidney.
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FIGURE 3. Urine osmolality following 12 h of water deprivation in mice with targeted disruptions of AT1 receptor subtypes. Open bar is AT1A/AT1B-deficient group, dark gray bar is AT1B receptor-deficient group, light grey bar is AT1A receptor-deficient group, and black bar is wild-type group *P < 0.0003 vs. wild-type or AT1B knockouts;  P < 0.0001 vs. wild-type or AT1B knockouts and P = 0.004 vs. AT1A knockouts. Figure reprinted from Ref. 12 with permission.
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Mice lacking AT2 receptors do not have overt abnormalities in urinary-concentrating function or in maintaining water balance. However, after a period of water deprivation their drinking response is blunted (7). Because exploratory behavior is globally diminished in AT2 knockout mice, the relative role of this behavioral abnormality and direct effects of the AT2 receptor on thirst mechanisms is not yet clear.
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Conclusions
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The study of AT1 receptor-deficient mice has reaffirmed and extended our understanding of the roles of AT1 receptors in regulating growth, development, blood pressure, and circulating volume. Gene-targeting experiments have also provided insights into novel functions of AT2 receptors.
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References
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Coffman T. Gene targeting in physiological investigations: studies of the renin-angiotensin system. Am J Physiol Renal Physiol 43: F999–F1005, 1998.
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Davisson R, Kim H, Krege J, Lager D, Smithies O, and Sigmund C. Complementation of reduced survival, hypotension, and renal abnormalities in angiotensinogen-deficient mice by the human renin and human angiotensinogen genes. J Clin Invest 99: 1258–1264, 1997.[Web of Science][Medline]
-
Esther C, Marino E, Howard T, Machaud A, Corvol P, Capecchi M, and Bernstein K. The critical role of tissue angiotensin-converting enzyme as revealed by gene targeting in mice. J Clin Invest 99: 2375–2385, 1997.[Web of Science][Medline]
-
Guyton A. Abnormal renal function and autoregulation in essential hypertension. Hypertension 18: 49–53, 1991.
-
Hall J. Control of sodium excretion by angiotensin II: intrarenal mechanisms and blood pressure regulation. Am J Physiol Regulatory Integrative Comp Physiol 250: R960–R972, 1986.[Abstract/Free Full Text]
-
Horiuchi M, Akishita M, and Dzau VJ. Recent progress in angiotensin II type 2 receptor research in the cardiovascular system. Hypertension 33: 613–621, 1999.[Abstract/Free Full Text]
-
Ichiki T, Labosky P, Shiota C, Okuyama S, Imagawa Y, Fogo A, Niimura F, Ichikawa I, Hogan B, and Inagam T. Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor. Nature 377: 748–750, 1995.[Medline]
-
Ito M, Oliverio M, Mannon P, Best C, Maeda N, Smithies O, and Coffman T. Regulation of blood pressure by the type 1A receptor for angiotensin II. Proc Natl Acad Sci USA 92: 3521–3525, 1995.[Abstract/Free Full Text]
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Matsusaka T, Nishimura H, Utsunomiya H, Kakuchi J, Niimura F, Inagami T, Fogo A, and Ichikawa I. Chimeric mice carrying "regional" targeted deletion of the angiotensin type 1A receptor gene: evidence against the role for local angiotensin in the in vivo feedback regulation of renin synthesis in juxtaglomerular cells. J Clin Invest 98: 1867–1877, 1996.[Web of Science][Medline]
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Miyazaki Y, Tsuchida S, Nishimura H, Pope J, Harris R, McKanna J, Inagami T, Hogan B, Fogo A, and Ichikawa I. Angiotensin induces the urinary peristaltic machinery during the perinatal period. J Clin Invest 102: 1489–1497, 1998.[Web of Science][Medline]
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Oliverio M, Delnomdedieu M, Best C, Li P, Morris M, Johnson A, Smithies O, and Coffman T. Abnormal water metabolism in mice lacking the AT1A receptor for angiotensin II. Am J Physiol Renal Physiol 278: F75–F82, 2000.[Abstract/Free Full Text]
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Oliverio M, Kim H, Ito M, Le T, Audoly L, Best C, Hiller S, Kluckman K, Maeda N, Smithies O, and Coffman T. Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II. Proc Natl Acad Sci USA 95: 15496–15501, 1998.[Abstract/Free Full Text]
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Siragy H, Inagami T, Ichiki T, and Carey R. Sustained hypersensitivity to angiotensin II and its mechanism in mice lacking the subtype-2 (AT2) angiotensin receptor. Proc Natl Acad Sci USA 96: 6506–6510, 1999.[Abstract/Free Full Text]
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Tsuchida S, Matsusaka T, Chen X, Okubo S, Niimura F, Nishimura H, Fogo A, Utsunomiya H, Inagami T, and Ichikawa I. Murine double nullizygotes of the angiotensin type 1A and 1B receptors duplicate severe abnormal phenotypes of angiotensinogen nullizygotes. J Clin Invest 101: 755–760, 1998.[Web of Science][Medline]
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Introduction
Gene targeting and the...
